A Fluorescent Sensor for Imaging Reversible Redox Cycles in Living Cells

Abstract

Life in aerobic environments requires organisms to maintain strict control over their internal redox status. At the cellular level, aerobic respiration poses a particularly unique challenge for living systems, as the energy-releasing reduction of oxygen to water generates partially reduced reactive oxygen species (ROS) intermediates that can exert widely divergent physiological and/or pathological effects. 1 Unregulated production of ROS results in oxidative stress, and subsequent buildup of free radical damage to proteins, lipids, and nucleic acids is connected to serious human diseases where age is a risk factor. 2,3 However, emerging evidence suggests that controlled production of one ROS in particular, hydrogen peroxide, can mediate cellular signal transduction through reversible oxidation and reduction of cysteine thiols and other redox-active groups. 4-8The complex, reversible oxidation biology of the cell and its broad implications in human health and disease provide motivation for developing new ways to study dynamic redox chemistry in living systems. In this regard, fluorescence imaging with redox-responsive chemosensors is a potentially powerful approach to probe various stages of oxidative signaling, stress, or repair in real time in living cells. Traditional fluorescent probes for redox activity, including dichlorodihydrofluorescein or dihydrorhodamine 123, are useful for cellular studies but can only respond irreversibly to a single initial oxidation event. 9-11 In contrast, fluorophores that can respond reversibly to changes in oxidation or reduction events would be much more valuable for visualizing cycles of redox signaling, stress, or repair and their dynamic interconversion. A few redox-sensitive fluorescent reporters based on protein 12,13 or peptide 14,15 scaffolds have been described, but no small molecules have been reported to date for imaging reversible oxidation and reduction events in living biological systems. We now present the synthesis, properties, and live-cell imaging applications of Redoxfluor-1 (RF1), a new type of fluorescent sensor for detecting reversible redox cycles in aqueous solution and in living cells. RF1 features a dual colorimetric/fluorimetric readout for oxidation-reduction events, a >50-fold fluorescence dynamic range, and visible wavelength excitation and emission profiles to minimize cellular damage and autofluorescence. In addition, RF1 can be loaded into living cells and image multiple, reversible cycles of oxidative stress and reductive repair.Our design strategy for fluorescence detection of reversible oxidation-reduction events is inspired by the extensive use of disulfides as redox resevoirs in biology. We anticipated that integrating this unit into a fluorescein scaffold would provide a small-molecule redox reporter with desirable optical properties and biological compatibility. Along these lines, the synthesis of RF1 proceeds in three steps as shown in Scheme 1. Lithiation of naphthalene in the presence of TMEDA and quenching with sulfur affords disulfid...

Possibilities and Challenges for Quantitative Optical Sensing of Hydrogen Peroxide

“…For example, reversible probes were developed [69][70][71][72][73] which can be oxidized and reduced in vivo enabling visualization of the cellular redox state. While this does not yield H 2 O 2 concentration data, it enables studies of the dynamic balance between oxidants and reductants.…”

“…However, the authors also state that this redox sensor can be re-oxidized even by air [71]. Another approach relies on the redox probe naphtopyran-benzothiazolium, which reacts with H 2 O 2 as well as bisulfite [139].…”

Fluorescent probes for nitric oxide and hydrogen peroxide in cell signaling

“…For example, installation of boronic esters at the 3′ and 6′ positions of a xanthenone fluoran core produces Peroxyfluor-1 (PF1), where the boronates force this platform to adopt a closed, colorless, and non-fluorescent lactone form [34] and other ROS bursts by ratiometric imaging, indicators that can be targeted to specific subcellular regions to study localized signals, reagents that can respond to multiple oxidation and reduction cycles to visualize redox signaling and/or stress dynamics, and agents that can push toward imaging in whole organisms. We have made initial progress in the development of small-molecule ratiometric probes [37] and reversible redox sensors [38]. Finally, although this review has focused specifically on NO and H 2 O 2 indicators, new fluorogenic reagents for peroxynitrite [39], hypochlorous acid [40], superoxide [41], highly reactive oxygen species [42], and global nitrative stress [43] have also been reported recently.…”

A Near-IR Reversible Fluorescent Probe Modulated by Selenium for Monitoring Peroxynitrite and Imaging in Living Cells

“…8 Thus, we anticipate widespread interest in a redoxreversible NIR fluorescent probe, which would exhibit much more value for visualizing cycles of redox signaling and stress caused by peroxynitrite. 9 Here we report a redox-responsive NIR fluorescent probe for continuous monitoring of ONOO À . ONOO À is modulated by cellular antioxidant defense systems, 1,10 in which selenium (Se) plays an important role as the active site of the antioxidant enzyme glutathione peroxidase (GPx).…”